TIRF Microscopy

Total internal reflection fluorescence microscopy (TIRFM) is a highly sensitive technique to perform functional investigations in living cells. The high signal to noise ratio and a resolution in z direction of usually 70-250 nm above the coverslip/water interface allows to visualize and to analyse vesicles transport and signalling events, as well as kinetic studies and single molecules detection. Physical background is the total reflection of a laser beam at the interface of glass and water and the resulting electromagnetic wave, the so-called evanescent field. The energy of the evanescent field decreases exponentially with the distance of the interface coverslip/water and allows exciting fluorochromes.

Total Internal Reflection Fluorescence (TIRF) Microscopy is a technique that only illuminates dye molecules near a surface. In this video, the pioneer of TIRF Microscopy describes what this technique is used for, explains the principles of the evanescent wave, gives many examples of different microscope configurations used in TIRF, and shows how polarized light TIRF can be used to image membrane orientation.

William Hughes works at the Garvan Institute of Medical Research, Sydney (Australia). In his Lab Head position he is interested in the causes of diabetes particularly looking at changes in exocytic behavior of pancreatic beta cells as well as fat and muscle cells. TIRF microscopy is predestined for researchers looking at cellular processes near the cytoplasmic membrane.

This webinar will illustrate results obtained by biochemical, Epifluorescence, TIRFM, Confocal and GSD techniques. Depending on the aim of experimental question, different imaging techniques deliver insights into varying aspects of intracellular pathways. To achieve "True-to-detail imaging" of the spatial arrangement of proteins and other biomolecules in cells, GSDIM achieves resolutions up to 20 nm in x and y direction – beyond the diffraction limit of light microscopy. But Super-resolution microscopy can be applied in the axial (z-) direction, too. A recent commercial implementation of the astigmatism approach will be discussed in more detail during this webinar.

Live cell imaging is one tool in the cell biologist's tool box. There are, however, a variety of technologies and methods to achieve this. In this webinar, three practicing researchers discuss: Their use of live cell imaging and how it has helped them achieve publishable results.

The main feature of total internal reflection fluorescence (TIRF) microscopy is the employment of an evanescent wave for the excitation of fluorophores instead of using direct light. A property of the evanescent wave, which arises from the glass/water or glass/specimen interface, is that its propagation in z-direction gradually degrades, limiting its penetration depth into the specimen to some hundred nanometers.

Due to their special role in organ function and the exchange of biological components some body cells developed certain polarization characteristics. These are reflected in differences of their plasma membrane composition. The essential and fascinating task of polarized protein transport in epithelial cells is to get the right protein into the right membrane.

Total internal reflection fluorescence (TIRF) is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.

The special feature of TIRF microscopy is the employment of an evanescent field for fluorophore excitation. Unlike standard widefield fluorescence illumination procedures with arc lamps, LEDs or lasers, the evanescent field only penetrates the specimen by about 100 nm starting from the coverslip/medium interface.

The need for data validation and accessibility has never been greater than it is today. We are inundated with information from a multitude of resources, but how can we easily evaluate the accuracy of that data? In the past, the peer review process provided this and was often run by publishers.

Application of TIRF microscopy (Total Internal Reflection Fluorescence) allows the visualization of structures at the apical surface of polarized epithelial cells that have been hidden in conventional fluorescence microscopy images. Hence, the approach reveals new insights into the composition of this characteristic cell pole that elucidate processes in apical protein trafficking.

Using TIRF microscopy, scientists have been able to take a closer look at intracellular transport processes with the example of the galactose-binding protein Galectin-3, which has been identified as a potential apical sorting receptor.